Hormonal therapies have led to great improvements in the survival of women with estrogen receptor (ER)-positive breast cancers. However, residual tumor cells often become resistant to anti-estrogen treatment resulting in recurrences that are frequently more aggressive than the original cancer. Current ER- targeted hormonal therapies include selective estrogen receptor modulators (e.g. tamoxifen, raloxifene), pure antagonists (e.g. fulvestrant) and aromatase inhibitors, all of which can result in resistance following prolonged/chronic use. In addition, women taking SERMs also experience an increased incidence of endometrial thickening/hyperplasia, polyps and cancer. Multiple mechanisms have been described yielding these deleterious effects; however, most recently the 7-transmembrane spanning G protein-coupled receptor GPER has been demonstrated to contribute to both hormonal resistance and off-target effects in the uterus. This conclusion is supported by the fact that anti-estrogens act as agonists for GPER and that GPER activates growth factor receptor pathways that are important in hormonal resistance. In our previous work, we have discovered and characterized novel selective ligands for GPER that do not bind ER? or ER?. To date however, there are no known ligands that exhibit the inverse selectivity. Towards this overall goal, we have identified a family of novel small molecules that are highly selective for ER? and ER? vs. GPER. As estrogen and current anti-estrogens cannot distinguish between ER?/? and GPER, our newly identified small molecule provides the opportunity to create novel ligands and therapeutic agents to selectively manipulate and target classical ERs. Our hypothesis is that through selected chemical modifications to this first generation ER?/?-selective compound, we will optimize the overall affinity, receptor selectivity and agonist/antagonist profile with the ultimate goal of creating a truly ER?-selective antagonist. The specific aims of this proposal are 1. To design and synthesize a suite of derivatives based on our highly ER?/?-selective scaffold; 2. To evaluate and prioritize these compounds in vitro for receptor binding, cellular activation/inhibition of rapid and genomic pathways, cell proliferation and toxicity and 3. To determine the ability of compounds to modulate estrogen-dependent physiology in vivo, particularly the anti- tumor properties of lead compounds in mice bearing ER-dependent and anti-estrogen-resistant xenograft, orthotopic and PDX tumors. The successful completion of these aims should result in a better understanding of the ligand selectivity of ER?, ER? and GPER, the identification of innovative compounds that provide novel pharmacological tools for the study of estrogen biology and (patho)physiology, and, with their successful application in the clinic, reductions of the development of anti-estrogen resistant recurrences of breast cancer and off-target effects in the endometrium, ultimately enhancing survival and the quality of life of women with breast cancer.